Other investigators have reported that epidermal growth factor (EGF) inhibits the induction of casein synthesis by mouse mammary tissue in vitro. However, since the circulating level of EGF increases during lactation, and since functional EGF receptors are retained by the lactating cells, it seemed unlikely to us that EGF is an inhibitor of mammary differentiation in vitro. The current studies demonstrate, in fact, that EGF inhibits the induction of casein synthesis in vitro only when insulin, an essential hormone, is present in the culture medium at pharmacological concentrations. We reported previously that EGF can substitute effectively for prolactin (P) in the induction of alpha-lactalbumin activity (assayed as lactose synthetase) in rat mammary explants cultured in the presence of insulin and glucocorticoid. The time courses of induction with EGF or P are similar. Both EGF and P also promote similar elevations in the accumulated level of alpha- lactalbumin mRNA. However, although P produces a large increase in the accumulated level of immunoprecipitable alpha- lactalbumin (determined with polyclonal antibody), the induction of immunoprecitable alpha-lactalbumin by EGF is less than 10% of that which occurs with P. It appears, then that EGF, like P, can support alpha-lactalbumin gene expression and the formation of enzymatically active alpha-lactalbumin, but that P is required post-translationally for the production of immuno-active alpha- lactalbumin. A less likely interpretation is that the alpha- lactalbumin activity induced by EGF corresponds to a protein distinct from the alpha-lactalbumin present in rat milk.